Method for loading drug on non-denatured human H ferritin

A ferritin, non-denaturing technology, applied in the field of ferritin-encapsulated drugs, can solve the problems of difficult industrial transformation, unstable ferritin-drug complex, long incubation time, etc.

Pending Publication Date: 2021-02-26
NANJING NAMOMEI TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the pH-mediated ferritin depolymerization / repolymerization loading system, because the drastic pH change will damage the protein structure, the ferritin-drug complex is unstable after drug loading, making it difficult to realize industrial transformation 9 ;Urea-mediated ferritin depolymerization / repolymerization system, long incubation time, large protein gradient dialysis loss, and most importantly, limited drug loading efficiency, which cannot meet clinical needs

Method used

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  • Method for loading drug on non-denatured human H ferritin
  • Method for loading drug on non-denatured human H ferritin
  • Method for loading drug on non-denatured human H ferritin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Structural Analysis of the Small Molecule Drug Doxorubicin (Dox) Channel on the HFn Protein Shell

[0063] In this embodiment, the following method steps are used to analyze the structure of the channel of the small molecule drug doxorubicin (Dox) on the HFn protein shell:

[0064] Step (1) Construction of HFn expression plasmid: After the DNA sequence of HFn (shown in SEQ ID No: 2, its amino acid sequence is shown in SEQ ID No: 3) through the whole gene synthesis (Generay, Shanghai), use NdeI and It was digested with BamHI restriction endonuclease and cloned into the Escherichia coli (E.coli) expression vector pET22b (+) plasmid (Novagen) with NdeI and BamHI restriction enzyme cutting sites, and the sequence was confirmed to be correct by DNA sequencing.

[0065] Step (2) expression and purification of HFn: the above-mentioned obtained plasmid is transferred into E.coli BL21 (TransGen) expression strain, and the Escherichia coli after transformation grows ove...

Embodiment 2

[0069] Example 2 Mutation verification of Dox drug loading channel on the surface of HFn protein

[0070]In order to verify the existence of Dox drug loading channel on the surface of HFn protein, we synthesized a series of mutants of HFn. Including the HFn C90S / C102S / C130S (SEQ ID No: 4) mutant demonstrating the existence of the channel, and Enlarged-HFn-1 (D92G) (SEQ ID No: 5), Enlarged-HFn-2 (R43G+R79G ) (SEQ ID No: 6) and Block-HFn(C90S) (SEQ ID No: 7) with closed channel. On the HFn protein shell, the residues in the 89-91 part can shift sideways, and Cys90 and Asp91 have almost no interaction with the surrounding residues or water molecules, and the flexibility is particularly high. The region between C90S and C102S of the C90S / C102S / C130S mutant forms a hydrogen bond network formed by adjacent residues and bound water molecules. This hydrogen bond network relatively fixes the conformation of residues 89-91, making 89 The -91 residue is very stable and inhibits the lat...

Embodiment 3

[0074] Example 3 Promotes the loading of small molecule drugs by controlling the temperature

[0075] Based on the analysis results of the ferritin structure, we speculate that the heating method can promote the further opening of the ferritin drug channel, which is more conducive to drug loading.

[0076] method:

[0077] 1. Temperature gradient experiment

[0078] 15% glycerol, 2 mg WT-HFn, 0.75 mg doxorubicin Dox, and 50 mM Trisbuffer were added to 1 mL reaction system to make up to 1 mL, pH 8.0. Incubate for 4 hours at 4°C, 25°C, 37°C, 42°C, 50°C, 60°C, 65°C, and 72°C, respectively. Centrifuge at 12000rpm for 10min at 4°C, take the supernatant, and send it to the desalting column Hiprep TM 26 / 10 Desalting treatment removed free doxorubicin, and obtained HFn-Dox ferritin doxorubicin samples. The concentrations of HFn and Dox in the HFn-Dox samples were determined to calculate the Dox loading and HFn protein yield.

[0079] 2. Time Gradient Experiment

[0080] 15% gly...

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Abstract

The invention belongs to the technical field of ferritin packaging drugs, and particularly discloses a method for loading drugs on non-denatured human H ferritin. Through HFn-small molecule drug crystal structure analysis and identification, the findings show that 12 drug channels exist on a protein shell of HFn, the key amino acid determining the channels is Asp89-Cys90-Asp91-Asp92, and meanwhile, amino acids of the small molecule drug channels has the property of temperature factor regulation and control. Based on the discovery, under the conditions that the ferritin structure is not changedand the ferritin is not denatured, by exploring different incubation temperatures and / or different incubation conditions, on the premise that the ferritin structure is kept complete, determination ofthe optimal condition of the HFn entrapping the drugs is achieved. By utilizing the obtained entrapment condition under the non-denaturation condition, the HFn can be conveniently, quickly, efficiently and sufficiently used for entrapment of different small molecular drugs.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to Chinese patent application CN2020106856800 filed on Jul. 16, 2020, and is incorporated by reference in its entirety. technical field [0003] The invention belongs to the technical field of ferritin-encapsulated medicines, and in particular relates to a method for loading medicines on non-denatured human H-ferritin. Background technique [0004] Ferritin is an iron storage protein that plays an important role in cellular iron homeostasis and anti-oxidation. Due to its unique structure of 24 subunits self-assembled, a protein shell that can be genetically and chemically modified to achieve additional functions, and a hollow cavity capable of encapsulating drugs, ferritin has become a promising drug delivery vehicle. 1 . Recent studies have shown that the receptor of human heavy-chain ferritin (Heavy-chain ferritin, HFn) is transferrin receptor 1 (Transferrin receptor1, TfR1), that i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/113A61K47/69A61K45/00A61K31/704A61K33/243A61K31/555A61K31/7088A61P35/00
CPCA61K31/555A61K31/704A61K31/7088A61K45/00A61K47/6949A61P35/00A61K33/243C07K5/1021
Inventor 孙国明尹雨芳陈向茹
Owner NANJING NAMOMEI TECH CO LTD
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